Medicinal substances should be produced at a constant specific activity level so that they can be delivered safely. For example, assays for biological molecules such as heparin have variability from batch to batch in terms of chain length, molecular weight, composition, degree of sulphation, etc. Other substances that are extracted from natural substances also need to be standardized. See for example, U.S. Pat. No. 7,575,886. One such substance is defibrotide. Defibrotide is a heterogeneous mixture of single-stranded polynucleotides of varying lengths that is extracted from mammalian organs.
There are assays available to evaluate the biological activity of defibrotide, including the fibrin plate test and the thromboelastographic recording of the euglobulin lysis time (Prino G. et al., Indagini preliminari sull'attivitfibrinolitica, nell'animale e nell'uomo, di una nuova sostanza presente in diversi organi animali, Simposio Internazionale: La ricerca scientifica nell'industria farmaceutica in Italia, Rome, 2-4 Oct. 1975-11 Farmaco, Ed. Prat.) (1969),24,552-561), the plasmin method (U.S. Pat. No. 7,338,777), and the euglobulin method (WO2013/190582). Although these methods are useful pharmaceutical manufacturing tools, all these methods, which are based on the pro-fibrinolytic properties of defibrotide, involve an assessment of defibrotide's activity on isolated proteins or enzymes.
Thus, there is a need in the art for novel methods to determine the biological activity of defibrotide in a cellular context that provides an accurate and reliable process for assessing the potency, e.g., by comparison with a reference defibrotide standard preparation, of new batches of defibrotide regardless of the manufacturing process used.